Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. Whereas most simian immunodeficiency viruses (SIVs) use Nef to overcome restriction by tetherin in their non-human primate hosts, HIV-1 Vpu and HIV-2 Env have evolved to serve this function in humans due to the absence of sequences in the cytoplasmic domain of human tetherin that confer susceptibility to Nef. We recently identified compensatory changes in the cytoplasmic tail of gp41 that restore resistance to tetherin in a nef-deleted strain of SIV that regained a pathogenic phenotype in rhesus macaques. These observations are analogous to the adaptation of HIV-2 Env for antagonism of human tetherin and imply that resistance to tetherin is important for lentiviral pathogenesis. We have now identified HIV-1 Nef alleles that are able to counteract restriction by human tetherin, underscoring the extraordinary plasticity of the primate lentiviruses in adapting to the tetherin proteins of their hosts and revealing a previously unappreciated role for Nef in HIV-1 infection. The studies outlined in this proposal build on this work to reveal the fundamental mechanisms of lentiviral resistance to tetherin and their role in immunodeficiency virus infection. Our first objective (Aim 1) is to define the molecular interactions and cellular trafficking pathways involved in tetherin antagonism by Nef, and the fate of tetherin (degradation versus sequestration) in SIV-infected cells. These studies will build on work by our group identifying Nef as the viral gene product of SIV that counteracts restriction by tetherin and will lead to a better understanding of the mechanisms of lentiviral resistance to tetherin. Our second objective (Aim 2) is define the genetic changes associated with the gain of anti-tetherin activity by HIV-1 Nef and the mechanism by which HIV-1 Nef counteracts human tetherin. These studies are important for determining how widespread anti-tetherin activity is for Nef alleles of primary HIV-1 isolates, the circumstances that lead to the gain of this function by HIV-1 Nef, and the mechanistic differences in tetherin antagonism by HIV-1 Nef versus SIV Nef. Our third objective (Aim 3) is to assess the natural variation in anti- tetherin activity for primary HIV-2 Ev proteins and to define the sequences in Env required for this activity. These studies will reveal the prevalence/range of anti-tetherin activity for primary HIV-2 Env proteins and the molecular adaptations in Env that contribute to this activity. By comparing the activity of Env alleles derived from individuals with progressive courses of HIV-2 infection to Env alleles derived from asymptomatic individuals who contain virus replication, these studies will reveal whether differences in tetherin antagonism are related to differences in HIV-2 pathogenesis.